Bilirubin is a degradation product of hemoglobin. It has been estimated that approximately 200-230 milligrams of bilirubin and its derivatives are formed each day in the normal human adult by the degradation of hemoglobin within the liver, spleen, and bone marrow.
In human body fluids such as bile and serum, bilirubin exists in two different forms, these forms commonly being referred to in the clinical literature as conjugated bilirubin, B.sub.c, and unconjugated bilirubin, B.sub.u. The total bilirubin content B.sub.T represents the sum of B.sub.u and B.sub.c.
Unconjugated bilirubin has a well-established molecular structure and constitutes the predominant portion of the total bilirubin content. For example, in normal human adult serum B.sub.u constitutes about 80 percent by weight of the total bilirubin content thereof. The molecular weight of B.sub.u is 584 and its molecular structure is as follows: ##STR1##
B.sub.c, believed to represent about 1 to 20 wt percent of the total bilirubin present in normal adult serum, is unstable in pure form and extremely difficult to isolate. Extensive study and reseach effort has been conducted to isolate this bilirubin component and to determine its molecular structure.
B.sub.c has traditionally been considered to represent the reaction product of B.sub.u esterified with sugar groups. However, owing to its instability, the specific molecular structure of any conjugated form of bilirubin had not been established until quite recently when a diconjugate B.sub.c species was separated from human bile and, for the first time, the molecular structure of a B.sub.c species was determined by the present inventor with the aid of co-workers. See paper entitled "Human Conjugated Bilirubin--Isolation, Biosynthesis And Molecular Characterization By Direct Spectroscopic Analyses", T. W. Wu et al., presented at the Americal Association for Clinical Chemistry 31st Annual Meeting in New Orleans, Louisiana, July 15-20, 1979. An abstract of this paper appears in Clinical Chemistry, Vol. 25, No. 6, p. 1137, June, 1979. Even more recently, the present inventor has isolated and identified small amounts of a substance having this same molecular structure in human serum. This diconjugate form of bilirubin has now been identified in both bile and serum. It was first determined to have a molecular structure as follows: ##STR2## where R.sub.1 =glucuronic acid and R.sub.2 =glucuronolactone or R.sub.1 =glucuronolactone and R.sub.2 =glucuronic acid
and since has been determined also to have a related diconjugate structure wherein both R.sub.1 and R.sub.2 are glucuronic acid. A monoconjugate species also exists wherein R.sub.1 is glucuronic acid or glucuronolactone and R.sub.2 is --OH. These mono- and diconjugate species coexist in any given body fluid sample and are, essentially, as far as can be ascertained, spectrally indistinguishable. Accordingly, a molecular weight assignable to B.sub.c falls within the range from about 750 to about 940. The singular term B.sub.c, therefore, as used herein represents a composite of B.sub.c species having a molecular weight in the above defined range.
The diagnostic significance of bilirubin is well established. For example, an excessive amount of bilirubin within the human body, referred to as jaundice, is recognized as evidence of a variety of disease conditions, particularly diseases of the liver. In addition, in certain pathological conditions, for example, obstructive jaundice, the small amount of B.sub.c normally present in adult human serum becomes elevated to form a larger proportion of the total bilirubin content. Thus, to facilitate early diagnosis of certain disease states, a bilirubin analysis that selectively determines the presence and/or concentration of both B.sub.c and B.sub.u, as well as the total bilirubin content of human serum, would be highly useful.
Prior to the present invention, to the knowledge of the inventor, no radiometric assay (i.e., no assay based on detection of spectral absorption or emission) was available for the selective determination of B.sub.c and B.sub.u. Various assays are available which provide so-called "direct" and "indirect", as well as "total" bilirubin values. Some authors have claimed that "direct" bilirubin values can be equated with B.sub.c, while the "indirect" values correspond to B.sub.u. However, as observed by Henry, Cannon, and Winkelman in Clinical Chemistry, Principles And Technics, Harper and Row, p. 1045 (1974), "direct" bilirubin samples have been found to include B.sub.c components, such as bilirubin diglucuronide, as well as B.sub.u. Thus, one cannot simply equate "direct" or "indirect" bilirubin values with either the conjugated or unconjugated bilirubin components of a biological liquid.
Furthermore, some believe that "direct" and "indirect" bilirubin have the same absorption spectra in serum. See Henry et al. referenced above at pp. 1071 and 1072. Based on this view, one would not expect that the different forms of bilirubin could be spectrophotometrically differentiated in a radiometric assay.
Prior to the present invention, radiometric assays for bilirubin, such as colorimetric and fluorimetric assays, measured an absorption or emission spectrum of bilirubin or a bilirubin reaction product and determined final bilirubin concentration values essentially on the basis that the total bilirubin present in an unknown sample was predominantly in the form of B.sub.u. For example, a colorimetric assay can be conducted by (i) detecting the absorbance, A, of an unknown bilirubin-containing sample; (ii) applying Beer's Law: EQU A=.epsilon..multidot.C.multidot.L III
where
A represents absorbance, PA0 .epsilon. represents molar absorptivity of bilirubin or a bilirubin reaction product PA0 C represents bilirubin concentration in moles/liter, and PA0 L represents pathlength;
and (iii) comparing the detected value of A to a calibration curve based on known amounts of B.sub.u ; whereby the molar concentration, C, of bilirubin in an unknown sample can be determined. The resultant molar concentration, C, is then converted to an absolute amount, such as mg/dl, using the molecular weight of B.sub.u.
These radiometric assays for bilirubin fail to account for the presence of B.sub.c and essentially ignore its contribution to the absorption and/or emission spectra of an unknown bilirubin-containing sample. To the extent that normal adult serum is composed predominantly of B.sub.u, the foregoing failure poses no real problem. However, in those cases where the concentration of B.sub.c is elevated so that it represents a larger than normal proportion of the total bilirubin content, the foregoing failure of known radiometric assays for bilirubin leads to serious assay error. Moreover, prior to the present invention, in the absence of molecular weight and spectral absorption and/or emission data on B.sub.c, the foregoing radiometric assay errors were difficult, if not impossible, to prevent.
Recently, Wu et al U.S. Pat. No. 4,069,017 issued Jan. 17, 1978, and Wu et al U.S. Ser. No. 932,158 filed Aug. 9, 1978, now U.S. Pat. No. 4,204,839, have described a new colorimetric and a new fluorimetric assay, respectively, for the determination of bilirubin. These new assays employ interactive mordants for bilirubin.
The mordanted bilirubin, as described in U.S. Pat. No. 4,069,017, facilitates the colorimetric detection of bilirubin in an aqueous liquid sample owing to the marked increase in the molar extinction coefficient exhibited by the mordanted bilirubin compared to that of free bilirubin and by the shift in absorption peak of the mordanted bilirubin compared to that exhibited by free bilirubin admixed in an aqueous liquid. The mordanted bilirubin, as described in U.S. Ser. No. 932,158, has also been found to exhibit fluorescence and therefore one can also determine the presence and/or concentration of bilirubin fluorimetrically by use of the mordanted bilirubin. Neither U.S. Ser. No. '158 nor U.S. Pat. No. '017, however, disclose how the mordant interacts with B.sub.u and B.sub.c individually.